In vitro assembly of outer membrane protein PhoE of E. coli.

Abstract

The cell envelope of Gram-negative bacteria, such as Escherichia coli, contains a double membrane. The inner membrane (IM) surrounds the cytoplasm and the outer membrane (OM) protects the cell by forming a barrier for harmful compounds, such as bile salts and antibiotics. The periplasm is located between the two membranes and contains the peptidoglycan. The OM is an asymmetric bilayer. The inner monolayer contains phospholipids and the outer monolayer contains lipopolysaccharides (LPS) (Lugtenberg and van Alphen, 1983). The OM contains various proteins (OMPs), including pore-forming proteins, which allow the passage of small hydrophilic molecules with molecular weights up to about 600 Da (Benz and Bauer, 1988). In contrast to inner membrane proteins, which span the membrane by hydrophilic a-helices, OMPs span the membrane by amphipathic B-strands. After translocation across the IM, OMPs have to fold and insert into the OM. These late steps in OMP biogenesis are the subject of our study. The porin PhoE, whose synthesis is induced when bacteria are grown under phosphate starvation (Overbeeke and Lugtenberg, 1980), is used as a model protein. The PhoE protein forms general diffusion pores with a selectivity for anions (Benz et al1985). The crystal structure of PhoE has been resolved (Cowan et al1992). Each monomer of this trimeric protein forms a 16-stranded antiparallel B-barrel with hydrophobic amino acid residues exposed to the lipids and to the subunit interface.